This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Brief anesthesia, such as following exposure to high levels of carbon dioxide, prior to decapitation is considered a more humane alternative for the euthanasia of rodents, compared to use of the decapitation alone. Studies of the levels of certain stress hormones in plasma such as corticosterone and adrenocorticotropic hormone (ACTH) have supported the use of this method of euthanasia in endocrinological and molecular studies. In the current study, rats were briefly exposed to a chamber filled with carbon dioxide until recumbent (20-25 s), and immediately sacrificed via decapitation, and trunk blood collected;findings were compared to rats sacrificed via decapitation with no exposure to carbon dioxide. Radioimmunassays were used to measure arginine vasopressin (AVP) and ACTH immunoreactivity (ir) in plasma. While ACTH-ir levels remained steady following brief exposure to carbon dioxide (in accordance with results of other investigators), AVP-ir levels were increased by more than an order of magnitude. These results were confirmed by quantitative capillary-liquid chromatography-mass spectrometry, indicating this observation of rapid increase in plasma AVP-ir levels is not due to nonspecific recognition by the antibody used in the radioimmunoassay. Likewise, using capillary-liquid chromatography-mass spectrometry, we observed a rapid increase in plasma oxytocin levels following carbon dioxide exposure. These surprising findings have important implications for the design and interpretation of studies involving brief carbon dioxide exposure prior to decapitation, as well as those with euthanasia resulting from carbon dioxide-induced asphyxiation. A manuscript describing this work is currently under review.